Sample 1 and 5 are the same sample, but 5 is diluted. Lanes 3 and 6 are the same, but 6 is also diluted. What steps can be taken so it is not degraded? I extracted and performed a clean up on the same day. I froze it and thawed it once to run the gel.

Do not over pipetting during your mixing with the dye to loading samples in the wells. Another thing, I can see some traces of nucleoproteins in the wells, which means that your genomic DNA is good but you need to clean it more by removing these proteins. You can do this, by the final wash using the 70% ethanol step. Either by using more time, due to the HMW, or the high concentration you get from each isolate tube. Another thing, dilute using a TE buffer, not water. And let it dissolve for at least 10 minutes on room temp. Before you store it in -80. If you are going to use the tube many times, dilute the master tube in many tubes to avoid multiple thaw/freez that can affect the % of degraded DNA in your tube. Every time you freez/thaw your tube, you degrade at least 10% of it.

Thanks for these great recommendations Ahmed. A few things: I have used phase lock tubes to separate my layers and transferred my upper layer twice. Should I spin and transfer into new phase lock tube a few more times to eliminate these proteins?

Also, when you said to use more time in the 70% ethanol step, are you referring to the centrifugation step that follows the addition of this? The protocol I have states to spin for 5mins.

One more note: I dissolved my pellet in 10mM Tris, 0.1mM EDTA, per protocol instructions.

does the tube has a white filter on it? if yes, such tubes are designed to minimize such proteins, however, sometimes it trap both the DNA and the attached proteins to the DNA altogether on the filter membrane. therefore, to get rid of these proteins, extra washing is preferred. 

yes as you did exactly, keep the centrifuge as it is for 5 min, but make the first wash step by using an 85% ethanol, and the 2nd wash as 75% but for 7-10 min. to wash the proteins and dry the membrane and to ensure better quality, leave the membrane that is now containing your cleaned HMW DNA to dry after the centrifuge for 5 min on room temperature to ensure that every and all traces of ethanol were evaporated. then, now you can add your TE buffer as you already done.  

The phase lock tubes have a white/cream-ish gel

I will follow your wash recommendations. I really appreciate this.

Goodluck;

I noticed that you said that you transfered the upper phase (containing your DNA) twice. 

May I ask you how? Because usually the upper phase volume is about 750 ul, and if we need to keep the HMW is intact we use a wide-pore puppet tips to avoid breaking the long strings of DNA. If we run out of these wide-pore pippet tips, we were using a sharp scluble to cut the pointed tip of a new 1000 ul puppete tip (usually blue colored) to make it wider opening.  and we use of course single wide pipite tip for each tube, which means you will need to prepare a nice amount of wide tips before you work. 

Hi Ahmed. I just used a regular pipette tip.

Well, I suggest to cut its tip to make it wider opening. And slowly suck the upper phase, or better, pour it off to a new tube by leaning the tube (containing the DNA upper phase) over the new tube. So that you can avoid the pipetting.By the way, your DNA in the picture is very nice for any PCR, why do you need to re-isolats it again? Just make a dilution and run a PCR.

Thank you Ahmed. You've been very helpful. My DNA needs to get sequenced. This was a trial run, just to see what I would expect when I worked with the actual samples. Glad I did this because everyone here offered very useful tips.

Wish you all the best :) 

Is this considered an acceptable, good quality DNA for sequencing? Note: the concentration for both samples were very low.

how much ul of DNA you loaded? i can say it is between 45-65 ng/ul for the clear one, while it is almost 30-40 ng for the second one because i can hardly see it.  anyhow, each company/lab requires its specific concentration. but in general, i send my sample as 50 ng/ul in a volume of 30 ul. so i think it is very good to be sent for seq. but you need to confirm with the lab. before you do so. you can send them the same picture and they will tell you if it is proper for their machine or they need higher conc. good luck 

Hi Ahmed,

Thanks for getting back to me so quick. If you recall from the first gel, I was asking if the gel was suppose to look the way it did. Since then, I was able to follow the suggestions on the thread. The first sample was a piece of rat tail washed with 70% ethanol. The second one is a piece from the same tail, but washed with 85% and then 75% ethanol, like you suggested. 

Noticeably, the first piece had a higher concentration than the second, before and after phenol chlroroform clean up.

To answer your question, I loaded 4ul. I know this concentration is very low to submit to the lab (they are requiring 5ug measured by nanodrop). This is another test run to see if I could resolve the issue seen in my first gel.

In both instances my concentration was low, so my next step will have to be to elute with a lower volume during my extraction.

the good result from your comment is that you are now narrowed the problem in one major reason. it is the wash step. So, again, the new info here is that the sample was a mouse tail. in this case, your problem is not in the wash. it is in the sample collection. or storing. besides, the tissue for mouse or rat is fatty, due to the skin, which means that, when you take the upper aquas phase containing your DNA from the phenol extraction buffer, you need to avoid the middle phase and the bottom ones. this will minimizing your total volume but  will rescue your DNA from the fatty molecules. another thing, your faint sample is very clean, which means that your treatmnet wash using the 85 was good, but, you need to do it with higher conc. of DNA. So, if the DNA is keep getting up as this low comc. then do not use the 85% wash. because it is sever for such conc. you have. Also, do not use long time exposing your DNA to phenol treatment. the phenol is degrading your DNA. another thing. how do you extarct or gring the tissue of the tail. this step is critical as well. the temp should not exceed 0C. which makes the grinding is very hard to soften the cell extract.

The tail is snipped and frozen immediately after collection. To extract I use Qiagen's Dneasy Blood and Tissue kit. I incubate the tail overnight at 56C to lyse, which dossolves the tissue, sort of speak.

The sample is exposed to the phenol for a little over 5 mins, and that is because according to the protocol I have, I have to spin for 5 mins after adding the phenol chloroform.